The present invention concerns compounds that inhibit cyclin-dependent kinases, particularly the cyclin-dependent kinase CDK4, and methods for treating cancers using such compounds.
Physiology
In a normal cell CDK4:cyclin D kinase holoenzyme phosphorylates the retinoblastoma protein (Rb) to form hyperphosphorylated retinoblastoma-phosphate (Rb-p). The hyperphosphorylation of retinoblastoma protein results in the release of Rb-p associated transcription factors that allow cell cycle progression beyond the G1 checkpoint, thereby promoting cell proliferation (Schrr et al., U.S. Pat. No. 5,723,313, (1998)).
The p16 gene (also known as CDKN2, MST1, and CDK4I) encodes the protein p16INK4A, which inhibits the cyclin-dependent kinase (CDK)4:cyclin D complex (Serrano, et al., Nature 366: 704-7 (1993)). Defects in the p16/CDK4:cyclinD/Rb pathway may lead to tumor formation. Genetic alteration or over expression of CDK4 and CyclinD1 has been observed in various tumor cell types. In addition, alterations of p16 have been described in various histologic types of human cancers including retinoblastoma, astrocytoma, melanoma, leukemia, breast cancer, head and neck squamous cell carcinoma, malignant mesothelioma, and lung cancer (Kamb et al., Science 264: 436-40 (1994); Noborie et al., Nature 368: 753-56 (1994); Walker et al., Cancer Res. 55: 20-3 (1995) and Nakagawa et al., Oncogene 11: 1843-51 (1995)).
Acridones and Benzothiadiazines
Acridones and benzothiadiazines (BTDs) are classes of known cyclic aryl compounds. Certain known acridones or BTDs have pharmacological effects. For example, BTDs have been investigated as diuretics (See de Tullio et al., J. Med. Chem). Fajans and Floyd (Ann. Rev. Med. 30:313-329, 1982) disclose the use of xe2x80x9cdiuretic benzothiadiazine, e.g. trichlormethiazidexe2x80x9d as a hyperglycemic in the treatment of insulinomas. Fajans and Floyd, however, do not teach the use of BTDs to affect cancers directly. The prior art, as understood, does not appear to teach the use of BTDs for their direct antineoplastic effect in the specific inhibition of CDK4 dependent tumors.
Particular acridones and acridines are known. For example, (C18H19N3O2xe2x80x94HCl) has been mentioned in a paper concerned with the anti-tumor activity of linear tri-cyclic carboxamides (Palmer et al., J. Med. Chem (US) 31 (4) pgs.707-721, 1988). Interestingly, the Palmer et al. paper states that this compound is xe2x80x9cinactivexe2x80x9d (page 711, column 1, paragraph 3).
The basic thioacridone ring structure was described in DeLeenheer et al., J. Pharm. Sci. 60:1238-1239, 1971, and is shown below. 
1-nitro-9-acridone, 1-nitro-10-(3-N,N-dimethylaminopropryl)-9-acridone, 1-amino-2,4-diethylthio-9-acridone and a number of acridine derivatives have been disclosed by Weltrowski et al. (Pol. J. Chem Technol. 56:77-82, 1982). This paper, however, deals exclusively with the synthesis of nitroacridines and does not discuss any biological activity or mechanism of biological action. But, the title of the Weltrowski article refers to tumor inhibition, and the footnote states that the work was supported by the Polish National Cancer Program.
The present invention concerns acridones, benzothiadiazines and derivatives thereof that are useful for treating cancers. The invention also concerns methods for using these compounds as CDK4 inhibitors to treat cancers.
There are a number of dreadful and relatively common cancers that have been shown to involve alterations in p16. These cancers include lung cancer, breast cancer, melanoma, leukemia, retinoblastoma, astrocytoma, head and neck squamous cell carcinoma and malignant mesothelioma. Expression of normal p16 protein in tumor cells with alterations of p16 results in restoration of cell-cycle regulation, decreased cell growth and decreased tumorigenicity in vivo. Because the only known function of p16 is inhibition of CDK4 kinase activity, cancers with alterations of p16, including those listed above, are likely to be sensitive to CDK4 inhibitors. Prior inhibitors of cyclin-dependent kinases, such as flavopiridole, staurosporin, and UCN-01, inhibit CDC2 and CDK2 as well as the intended target, CDK4. This lack of specificity produces pathological side effects, such as bone marrow and gastrointestinal toxicities, and limits their clinical application.
As a result, there is a need for drugs for treating CDK4 sensitive neoplasms that minimize toxic side effects caused by concomitant inhibition of CDC2 and CDK2. The compounds claimed in this application inhibit CDK4 to a far greater extent than CDC2 or CDK2 and therefore satisfy this need.
One example of a novel compound of the present invention is 3-amino-9-thio(10H)-acridone. This compound and others can be used to form therapeutic compositions. One embodiment of such a composition comprises a therapeutically effective amount of a compound selected from the group consisting of a benzothiadiazine, a thioacridone, or mixtures thereof. The compound has an IC50 for CDK4 of less than about 10 xcexcM, preferably from about 1 xcexcM to about 7 xcexcM, an IC50 for CDC2 of greater than about 60 xcexcM, preferably greater than about 100 xcexcM, an IC50 for CDK2/A of greater than about 100 xcexcM, an IC50 for CDK2/E of greater than about 80 xcexcM, and preferably greater than about 100 xcexcM.
The specificity of the compounds for inhibiting CDK4 can be expressed as a ratio of the IC50 values for other enzymes relative to CDK4. Such compositions typically comprise a compound selected from the group consisting of a benzothiadiazine, a thioacridone, or mixtures thereof, the compound having an IC50 ratio for CDC2:CDK4 of greater than about 8.5, typcially greater than about 20, preferably greater than about 60; an IC50 ratio for CDK2/A:CDK4 of greater than about 14, typically greater than about 20, and preferably greater than about 60; and an IC50 ratio for CDC2/E:CDK4 of greater than about 11.5, typically greater than about 20, and preferably greater than about 60.
The invention also provides a composition comprising an effective amount of a compound according to Formula 1 
where m is 0 or 1, n=m, R1xe2x80x94R4 are independently selected from the group consisting of H, xe2x80x94NH2 and lower alkoxy, where with m=1 one of R1xe2x80x94R4 is an amine bonded to Rxe2x80x2 to form an arylamide, or Formula 2 
where R and R1 are independently carbon or nitrogen, where if R1=carbon X is hydrogen, halogen, aryl or alkoxy, and R2 is selected from the group consisting of lower alkyl and aryl amino. The composition also can comprise mixtures of compounds satisfying Formula 1 and/or Formula 2. The composition can further include, without limitation, additives selected from the group consisting of carriers, diluents, excipients, diagnostics, direct compression buffers, buffers, stabilizers, fillers, disintegrates, flavors, colors, and mixtures thereof.
A method for inhibiting the growth of living cells also is described. The method comprises providing a compound selected from the group consisting of a benzothiadiazine, a thioacridone, or mixtures thereof, as described above. An effective amount of the compound, a mixture of compounds, or a composition comprising the compound or mixture of compounds, is administered to a subject to inhibit the growth of living cells.